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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.133, #1)

Role of selenium supplementation and heat stress on trehalose and glutathione content in Saccharomyces cerevisiae by Sukesh Chander Sharma; Maninder Singh Anand (pp. 1-7).

The role of selenium (Se) supplementation on glutathione, a potent intracellular redox buffer, and trehalose, a well-known stress protectant molecule, was studied. The amount of glutathione decreased significantly while that of trehalose showed a minor decrease in the cells grown in Se-supplemented medium. After heat shock, glutathione content diminished further, whereas that of trehalose increased significantly in control and Se-supplemented cells. These findings suggest the importance of trehalose as an anti-oxidant molecule.

Keywords: Antioxidants; heat shock; oxidative stress; selenium supplementation; yeast

Adenoviral vectors by Siddhartha S. Ghosh; P. Gopinath; A. Ramesh (pp. 9-29).

Gene therapy is a promising tool for treatment of the human diseases that cannot be cured by rational therapies, and its primary success depends on suitable vectors to deliver therapeutic genes. Adenoviruses (Ads) are among the most commonly used vectors for gene therapy, second only to retroviruses. During the last decade, remarkable progress has been made in the development of Ad vectors and in the understanding of the toxicity related to the Ad vector system. Ad vector has certain advantages such as high transduction efficiency for different quiescent and dividing cell types and high levels of short-term expression to provide therapeutic benefits. However, researchers are facing the challenges associated with tissue-specific targeting of vectors and the vector-mediated immunogenicity. This review mainly focuses on the studies that have employed methods to improve Ad vectors and reduce viral toxicity for different applications. These methods include minimization or elimination of viral genes, retargeting of vector to the tissue of interest, and generation of immunocompromised recombinant vectors that lead to safer use of Ad vector systems that improve persistence of transgene expression. Moreover, the therapeutic applications of Ad vectors for liver-targeted gene therapy, suicide gene therapy, delivery of small interfering RNA, and production of recombinant vaccine under regulated conditions used in clinical trials are discussed.

Keywords: Adenovirus vector; inverted terminal repeat; small interfering RNA; Coxsackie and adenovirus receptor

Enzymatic synthesis of prebiotic oligosaccharides by Maria C. Rabelo; Talita L. Honorato; Luciana R. B. Gonçalves; Gustavo A. S. Pinto; Sueli Rodrigues (pp. 31-40).

Prebiotic oligosaccharides are nondigestible carbohydrates that can be obtained by enzymatic synthesis. Glucosyltransferases can be used to produce these carbohydrate through an acceptor reaction synthesis. When maltose is the acceptor a trisaccharide composed of one maltose unit and one glucose unit linked by an α-1,6-glycosidic bond (panose) is obtained as the primer product of the dextransucrase acceptor reaction. In this work, panose enzymatic synthesis was evaluated by a central composite experimental design in which maltose and sucrose concentration were varied in a wide range of maltose/sucrose ratios in a batch reactor system. A partially purified enzyme was used in order to reduce the process costs, because enzyme purification is one of the most expensive steps in enzymatic synthesis. Even using high maltose/sucrose ratios, dextran and higher-oligosaccharide formation were not avoided. The results showed that intermediate concentrations of sucrose and high maltose concentration resulted in high panose productivity with low dextran and higher-oligosaccharide productivity.

Keywords: Enzymatic synthesis; optimization; factorial design; surface response analysis; prebiotic oligosaccharides

Pretreatment of corn stover by low-liquid ammonia recycle percolation process by Tae Hyun Kim; Yoon Y. Lee; Changshin Sunwoo; Jun Seok Kim (pp. 41-57).

A pretreatment method using aqueous ammonia was investigated with the intent of minimizing the liquid throughput. This process uses a flow-through packed column reactor (or percolation reactor). In comparison to the ammonia recycle percolation (ARP) process developed previously in our laboratory, this process significantly reduces the liquid throughput to one reactor void volume in packed bed (2.0–4.7 mL of liquid/g of corn stover) and, thus, is termed low-liquid ARP (LLARP). In addition to attaining short residence time and reduced energy input, this process achieves 59–70% of lignin removal and 48–57% of xylan retention. With optimum operation of the LLARP to corn stover, enzymatic digestibilities of 95, 90 and 86% were achieved with 60, 15, and 7.5 filter paper units/g of glucan, respectively. In the simultaneous saccharification and fermentation test of the LLARP samples using Saccharomyces cerevisiae (NREL-D₅A), an ethanol yield of 84% of the theoretical maximum was achieved with 6% (w/v) glucan loading. In the simultaneous saccharification and cofermentation (SSCF) test using recombinant Escherichia coli (KO11), both the glucan and xylan in the solid were effectively utilized, giving an overall ethanol yield of 109% of the theoretical maximum based on glucan, a clear indication that the xylan content was converted into ethanol. The xylooligomers existing in the LLARP effluent were not effectively hydrolyzed by cellulase enzyme, achieving only 60% of digestibility. SSCF of the treated corn stover was severely hampered when the substrate was supplemented with the LLARP effluent, giving only 56% the overall yield of ethanol. The effluent appears to significantly inhibit cellulase and microbial activities.

Keywords: Corn stover; pretreatment; aqueous ammonia; bioenergy; simultaneous saccharification and cofermentation

Inactivation of Botrytis cinerea during thermophilic composting of greenhouse tomato plant residues by A. E. Ghaly; F. Alkoaik; A. Snow (pp. 59-75).

The effectiveness of in-vessel thermophilic composting on the inactivation of Botrytis cinerea was evaluated. The bioreactor operated on an infected mixture of tomato plant residues, wood shavings, and municipal solid compost (1∶1.5∶0.28). Tap water and urea were added to adjust the moisture content and C∶N ratio to 60% and 30∶1, respectively. Used cooking oil was added as a bioavailable carbon source to compensate for heat losses from the system and extend the thermophilic compositing stage. The controlled thermophilic composting process was successful in inactivating B. cinerea. During all experiments, the average reactor temperature increased gradually, reaching its peak after 31 h of operation. Temperatures in the range of 62.6–63.9°C were maintained during the thermophilic stage by the intermittent addition of used cooking oil. The results of the enzyme-linked immunosorbent assay test indicated that the initial concentration of B. cinerea in the compost samples (14.6 μg of dried mycelium/g of compost) was reduced to 12.9, 8.8, and 2.4 μg/g after 24, 48, and 72 h of thermophilic composting, respectively. Plating assay indicated that the mold was completely inactivated in samples after 48 h of thermophilic composting. No significant reduction in B. cinerea was observed during the transient phase (first 30 h of rising temperature) because the temperature reached the lethal level of 55°C after 23 h, thus allowing only 7 h of exposure to temperatures higher than 55°C during this phase. The relatively short time required for complete inactivation of B. cinerea was achieved by maintaining a constant high temperature and a uniform distribution of temperature and extending the duration of the thermophilic stage by the addition of the proper amount of bioavailable carbon (used cooking oil).

Keywords: Compost; temperature; inactivation; tomato remains; Botrytis cinerea

Expression and purification of a mutant of human interleukin-2 in Pichia pastoris by Yan Liu; Xun-Yan Xiao; Min Sun; Ying-He Hu; Ke-Qing Ou-Yang; Shao-Xi Cai; Zi-Chun Hua (pp. 77-86).

Interleukin (IL)-2 is a pharmacologically important cytokine secreted by T-lymphocytes. Recombinant IL-2 (rIL-2) has been modified and produced in many systems. Mass production of rIL-2 is the prerequisite for its wide application. Using a site-directed mutagenesis strategy, we first generated a gene coding for a new type of mutant of human IL-2 (MhIL-2), in which we replaced the cysteine-125 in human IL-2 with alanine, the leucine-18 with methionine, and the leucine-19 with serine. Then we investigated the possibility of its production of MhIL-2 in a Pichia pastoris system. High-level secreted expression of MhIL-2 was achieved by methanol induction. When purified with ultrafiltration, cation-exchange chromatography, and Sephadex G100 gel filtration, about 100 mg of MhIL-2 with high purity was obtained from 1 L of ferment supernatant. Biologic activity assay revealed that the purified recombinant protein displayed increased activity on proliferation of IL-2-dependent CTLL-2 cells. These results suggest that MhIL-2 is an improved IL-2 mutant that might hold great promise for clinical use, and that P. pastoris is an excellent system for the mass production of biologically active hIL-2.

Keywords: Human interleukin-2; site-directed mutagenesis; Pichia pastoris ; purification; increased activity

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.133, #1)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.133, #1)